The present invention generally relates to liquid chromatography and more particularly to a polymer packing material for liquid chromatography and a producing method thereof. More particularly, the present invention relates to a producing method of a polymer packing material according to a two-step or multiple step polymerization process, as well as to a polymer packing material produced according to such a process.
The packing material for high performance liquid chromatography (HPLC) is generally classified into an inorganic packing material that uses an inorganic carrier and a polymer packing material that uses an organic polymer.
In actual use, inorganic packing materials based on silica gel are used most frequently. In a reversed phase liquid chromatography, which occupies more than 60% of the separation mode of HPLC, an alkylsiliation silica gel is used primarily, in which the surface of the silica gel carrier is chemically modified.
While such conventional inorganic packing materials show an excellent separation performance and mechanical strength, there has been a drawback in the conventional packing material such as low chemical stability or appearance of undesirable secondary retention effect caused by a silanol group remaining on the silica gel surface or by a metallic impurity contained in the silica gel base.
On the other hand, the polymer packing materials have an advantageous feature of chemical stability and have been used for a packing material of size exclusion chromatography or ion exchange chromatography similarly to a silica gel packing material.
The polymer packing materials are used also in a reversed phase liquid chromatography, particularly under a separation condition in which the use of silica gel packing material is not possible. The understanding on the separation characteristic of such organic polymer packing material is increasing, and there are reports claiming that a separation characteristic superior to the separation characteristic of a silica gel packing material is obtained.
A polymer packing material is generally classified into those prepared from a natural polymer by a cross-linking process, and those synthesized by a polymerization process of a vinyl polymer.
The representative example of the former includes a packing material derived from a polysaccharide derivative such as agarose, dextran or mannan. These materials, however, generally suffers from the problem of low withstand pressure and may not be used for the packing material of HPLC.
On the other hand, the synthetic polymer packing material includes materials such as a polystyrene-divinylbenzen gel, a derivative thereof, a poly(meta)acrylate gel and a polyacrylamide gel. Among others, the polyalkylmetacrylate gel or the polystyrene-divinylbenzene gel is chemically stable and used for the packing material in the reversed phase liquid chromatography. It should be noted that these gels are stable over a wide pH range as compared with the silica gel packing material.
Such a synthetic organic polymer packing material is generally synthesized by mixing monomers together with a diluent and a cross-linking agent to cause a polymerization such that a porous structure is formed.
Thereby, fine pores are formed when a good solvent is used for the polymer to be synthesized, while large pores tend to be formed when a poor solvent is used. In other words, the pore diameter is controlled by choosing the combination of the diluent and the monomers. By using such a procedure, spherical porous polymer particles are synthesized as the polymer packing material.
On the other hand, it is known also that there are problems in such synthetic polymer packing materials. One of the problems is related to the structure of the packing material, particularly with regard to the fine pores. The other problem is related to the size distribution of the porous polymer particles forming the packing material.
More specifically, a polymer packing material generally has a double pore structure called micropore, which arises in relation to the cross-linking structure of the polymer, wherein such a micropore generally has a diameter of smaller than 2 nm.
Because of the existence of the micropores in the porous polymer particles, the extent a solute molecule can penetrate by osmosis becomes larger for small solute molecules than for a polymer solute.
Accordingly, such a polymer packing material shows a separation characteristic substantially different from that of the silica gel packing material when used for separation of a specimen in a chromatograph column. As the effect of such micropores on the resolution is not fully understood, and in view of the fact that a complete control of the micropores is difficult, the use of the polymer packing material has frequently led to a deterioration of resolution of chromatography.
When the micropores could be eliminated, the separation characteristic of a polymer packing material would be improved substantially. By eliminating the micropores, it is expected that the separation characteristic of the polymer packing material is controlled as desired. Further, the type of the specimens to which the polymer packing material is applicable is expected to increase. Further, the resolution of chromatography is expected to increase.
About the problem related to the particle size distribution of the polymer packing material, the problem arises mainly as a result of the process employed for producing the polymer packing material.
As noted previously, the polymer packing materials are produced by a suspension polymerization process. However, the polymer packing materials obtained according to such a conventional process generally have a wide spectrum of particle size, and the desired column performance is not obtained when the as-formed polymer particles are filled directly in a chromatograph column as the polymer packing material.
In order to obtain a desired column performance, it has been practiced to subject the obtained polymer particles to a classification process so as to select those polymer particles having a diameter falling in a desired range.
However, such a classification process requires a specially built facility and the cost of the packing material is increased inevitably. In addition, the classification process uses only a part of the obtained polymer particles, while the rest of the polymer particles are discarded. Thus, the yield of the polymer packing material decreases, and this also contributes to the increase of the cost of the polymer packing material.
In order to eliminate the problem of the size distribution of the polymer packing material, there is proposed a so-called two-stage polymerization process for producing the polymer packing material.
FIG. 1 shows the process of producing a polymer packing material according to such a two-stage polymerization process.
Referring to FIG. 1, seed particles 101 having a uniform particle diameter of about 1 .mu.m are swelled by consecutively applying thereto a swelling agent in a first step (1) and a monomer in a second step (2), and the monomers in the swelled particles thus obtained are subjected to a polymerization process under heat in a third step (3) to form polymer particles 102 having a uniform diameter.
According to such a two-stage process, the uniformity of the particle diameter is maintained during the swelling process of the steps (1) and (2). Further, the polymer particles 102 are synthesized as highly polymerized particles. By using a diluent in the step (2) together with the monomers not reacting with the diluent and by removing the diluent in the polymerization process, the polymer particles 102 are formed to have a porous structure after the polymerization process.
It should be noted that the foregoing two-stage process can be carried out by a facility used conventionally for a suspension polymerization process, except for the swelling process. Thus, the desired mono dispersion particle distribution is obtained easily and with low cost for the polymer packing material.
It should be noted that the polymer particles forming the polymer packing material show a mono dispersion in the particle distribution in conformity to the particle distribution of the seed particles 101, and the classification process can be eliminated entirely. Further, the polymer packing material thus obtained show an excellent separation performance when used in a chromatograph column. In addition, the pressure loss of the polymer packing material is small and the polymer packing material can be filled stably in the chromatograph column. In addition, the separation using such a polymer packing material can be achieved at high speed.
However, such polymer packing material, although produced according to such a two-stage process, still suffers from the problem of micropores similarly to the conventional polymer packing material produced by the suspension polymerization process.
Thus, the problem of the micropores remains unsolved and hence the removal of the micropores remains as an important target of research of polymer packing material.